This assay has high sensitivity and excellent specificity for detection of mouse NTX.

Cross-Reactivity (Details)

Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between the target antigen and all analogues for other species. Therefore, cross reaction may still exist.

Sensitivity

The sensitivity of this assay, or Lower Limit of Detection (LLD), was defined as the lowest protein concentration that could be differentiated from zero. It was determined as the mean O.D value of 20 replicates of the zero standard added by their three standard deviations.

Material not included

Microplate reader capable of measuring absorbance at 450nm, with the correction wavelength set at 540nm or 570nm.

An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.

Squirt bottle, manifold dispenser or automated microplate washer.

Absorbent paper for blotting the microtiter plate.

100mL and 500mL graduated cylinders.

Deionized or distilled water.

Pipettes and pipette tips.

Test tubes for dilution.

Application Details

Application Details

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Application Notes

The supplier is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.

Samples to be used within 5 days may be stored at 2-8°C, otherwise samples must be stored at -20°C (≤ 1 month) or -80°C (≤ 2 months) to avoid loss of bioactivity and contamination.

Grossly hemolyzed samples are not suitable for use in this assay.

If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.

Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.

Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.

Owing to the possibility of mismatching between antigens from another resource and antibodies used in this supplier's kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by this supplier's products.

Influenced by factors including cell viability, cell number and cell sampling time, samples from cell culture supernatant may not be recognized by the kit.

Fresh samples without long time storage are recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

Comment

Detection wavelength: 450 nm

Information on standard material:Some standards are from natural resource, some are recombinant protein, but the recombinant protein will not expressed from Baculovirus. Generally it's CHO cells, but that depends.The formulation of auxiliary material in the standard is classified, but it dosen't contain any poisonous substance. Proclin 300 (1:3000) is used as preservative.

Information on reagents:Most of the stop solution are 1 N H2SO4, a few is not. The formulation of wash solution is classified. None of the components contains (sodium) azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials. Some could contain BSA. For the sandwich method kits, the sample diluent, antibody diluent, enzyme diluent and standard all contain BSA.

Information on antibodies:The provided antibodies and their host vary in different kits. Some are affinity purified, some are Protein A or G purified

Sample Volume

50-100 μL

Assay Time

1 - 4.5 h

Plate

Pre-coated,Strips (12 x 8)

Protocol

This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for NTX has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any NTX present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for NTX is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of NTX bound in the initial step. The color development is stopped and the intensity of the color is measured.

Sample Collection

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2-8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.

Tissue Homogenates: Rinse 100mg tissue with 1× PBS, homogenize in 1mL of 1× PBS and store overnight at -20°C. After two freeze-thaw cycles to break the cell membranes, centrifuge the homogenates for 5 minutes at 5000 × g, 2-8°C. Remove and assay the supernate immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

Assay Precision

Intra-assay precision (precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess precision.Inter-assay precision (precision between assays): Three samples of known concentration were tested in twenty assays to assess precision.

Handling

Handling

The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.

Handling Advice

The kit should not be used beyond the expiration date on the kit label.

Do not mix or substitute reagents with those from other lots or sources.

If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.

Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/temperature and kit age can cause variation in binding.

This assay is designed to eliminate interference by soluble receptors, binding proteins and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage

4 °C/-20 °C

Storage Comment

May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.